a Heart organoid (HO) tracking over a time course (over n = 84, over six independent experiments). The cardiac-crescent-like structures (CCL, top) or sphere with buds (Sb; similar to a small heart tube with polarity, bottom) appeared within 3 days of culture. Arrows indicate buds in a Sb. These structures underwent continuous morphological changes into heart tubes (HT), looping heart tubes (LHT) and chambered hearts (scale bar: 100 µm). The HOs were classified as being in the cardiac-crescent-like, Sb, heart tube, looping heart tube, or chamber-formation (CF) stage. b Half of the cultured HOs showed spontaneous beating movement within 3 days of culture (57.6%). The number of beating HOs increased at day 6 (75.4%; n = 84, six independent experiments). See also Supplementary Movie 4. c Morphological changes in the EBs appeared within 3 days of culture. At day 3, EBs with a spherical shape prior to culture had transformed into cardiac-crescent-like structures or Sbs (92.0%). Most EBs showed morphological changes until day 6 (97.3%). d Frequency of HOs with certain morphological characteristics according to the culture period. The majority of the HOs were classified as being in the cardiac-crescent-like stage (red bar) at day 3 and transformed to the heart tube and looping heart tube stages (light green and purple, respectively, days 5−6), looping heart tube and chamber-formation stages (purple and black, respectively, day 9) and finally to the chamber-formation stage (black) at days 10−15. Data are presented as mean values ± SEM in (b–d).